transduction was investigated. Superoxide radical anion appears to be an upstream signal mediating the phorbol acetate (TPA)-stimulated transcriptional activation of the human Mn(II) superoxide dismutase (hMnSOD) gene. We identified the TPA-responsive element for TPA-induced hMnSOD mRNA, TGACGTCT (termed MSTRE), and the MSTRE binding protein, which is closely related to CREB-1/ATF-1 in A549 cells. The TPA-mediated induction of hMnSOD mRNA requires the MSTRE-CREB-1/ATF-1-like complex to be phosphorylated by PKC. This induction is blocked by inhibitors of NADPH oxidase, which catalyze superoxide radical anion generation. These results indicate that superoxide radical anion is a signal in inducing MnSOD. In another experiment, we studied growth factor-induced production of reactive oxygen species in cells and an elevation of tyrosine-phosphorylated proteins. This elevation can be achieved by the activation of protein-tyrosine kinases (PTKs) and/or inactivation of protein-tyrosine phosphatases (PTPs). Our results showed that superoxide radical anion is kinetically more efficient (about ten folds) and chemically more specific (reversibility) than hydrogen peroxide in inactivating PTP-1B. The initial oxidized product, the C-215 sulfenic derivative, can easily be oxidized further to its irreversible sulfinic and sulfonic derivatives. Glutathionylation of the sulfenic derivative to form S-glutathionylated PTP-1B, which can be reactivated by thioltransferase, prevents this irreversible inactivation of PTP-1B. This mechanism is supported by the in vivo study showing the formation of glutathionylated PTP-1B at C215 in A431 cells when treated with EGF. Together, our results indicate that superoxide radical anion is a second messenger in signal transduction.